Immune memory is critical for pathogen resistance as well as for vaccine efficacy; it is a defining feature of adaptive immunity. To deepen our fundamental understanding of how immune memory works we have fo- cused on elucidating the inherent differences between memory B cells (MBC) and nave B cells (NBC). A key limitation in studying murine MBC is their vanishingly small numbers and the difficulty in identifying and purify- ing them; hence, studies on function and gene expression have been quite limited. To overcome this barrier our lab has developed multiple systems by which to obtain and purify large numbers of homogeneous, bona fide MBC, allowing us to assay them in vivo and in vitro. In the last period we used this unique capability to de- fine subsets of murine MBC, revealed by expression of CD80, PD-L2, and CD73. We focused on the three subsets defined by CD80 and PD-L2: ?double positive? (DP), PD-L2-?single positive? (SP), and ?double nega- tive? (DN). Strikingly, upon reimmunization DP MBC are potent in generating AFCs but cannot generate GCs, whereas DN MBC make GC but lag at making AFCs; SPs are intermediate. This heterogeneity is present with- in IgM MBC or IgG MBC compartments and hence subset identity rather than isotype controls function. This finding was exciting because it explained how MBC could provide rapid effector function while still reseeding the memory compartment for future responses. It also revealed that studying unseparated mixtures of MBC could yield misleading conclusions. Our long-term goal is to understand the mechanisms that underlie this dif- ferent behavior among the MBC subsets and also between MBC and NBC overall. We will address this by taking both functional and genetic approaches. We recently published microarray- based data on mRNA expression of the MBC subsets, a good start in terms of defining the reprogramming that underlies differential functions. Along with our recently published functional data, this work forms the basis for our proposal. Despite progress, we have as yet only a rudimentary idea of functional differences among MBC subsets. Similarly, we have an incomplete catalog of intrinsic gene expression differences. And critical epige- netic differences have yet to be investigated. In Aim 1 we will delineate functional differences among MBC subset cells both in vivo and in vitro, starting with single cell RNA-seq to discover hidden additional heteroge- neity. In Aim 2 we will delineate intrinsic gene expression and epigenetic differences among MBC subsets and NBC. We expect from this analysis to determine the mechanisms that underlie the functional differences among these related cell types. In Aim 3 we will follow up our recent microarray data that identified transcrip- tion factors that are differentially expressed among MBC subsets; these are candidates to explain the different behaviors we have documented. We will delete two of these genes, Zbtb32 and Klf2, in established MBC, us- ing our B cell-specific inducible Cre mouse, and then test the functional consequences. Together these three Aims will thus both generate and test hypotheses, thereby providing new insights into B cell memory.